Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase.

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.

A lysine-cysteine redox switch with an NOS bridge regulates enzyme function.

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function1-4. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts5,6. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes7,8. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch9,10.

Lysines and cysteines: partners in stress?

Modifications of cysteine residues in redox-sensitive proteins are key to redox signaling and stress response in all organisms. A novel type of redox switch was recently discovered that comprises lysine and cysteine residues covalently linked by an nitrogen-oxygen-sulfur (NOS) bridge. Here, we discuss chemical and biological implications of this discovery.

Low-barrier hydrogen bonds in enzyme cooperativity.

The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins, with haemoglobin and aspartate transcarbamoylase serving as prototypical examples1,2. The binding of effectors typically causes a structural transition of the protein that is propagated through signalling pathways to remote sites and involves marked changes on the tertiary and sometimes even the quaternary level1-5. However, the origin of these signals and the molecular mechanism of long-range signalling at an atomic level remain unclear5-8. The different spatial scales and timescales in signalling pathways render experimental observation challenging; in particular, the positions and movement of mobile protons cannot be visualized by current methods of structural analysis. Here we report the experimental observation of fluctuating low-barrier hydrogen bonds as switching elements in cooperativity pathways of multimeric enzymes. We have observed these low-barrier hydrogen bonds in ultra-high-resolution X-ray crystallographic structures of two multimeric enzymes, and have validated their assignment using computational calculations. Catalytic events at the active sites switch between low-barrier hydrogen bonds and ordinary hydrogen bonds in a circuit that consists of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons by behaving as an atomistic Newton's cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide several lines of evidence and a working model for not only the existence of low-barrier hydrogen bonds in proteins, but also a connection to enzyme cooperativity. This finding suggests new principles of drug and enzyme design, in which sequences of residues can be purposefully included to enable long-range communication and thus the regulation of engineered biomolecules.

Widespread occurrence of covalent lysine-cysteine redox switches in proteins.

We recently reported the discovery of a lysine-cysteine redox switch in proteins with a covalent nitrogen-oxygen-sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur-oxygen-nitrogen-oxygen-sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors, linking lysine chemistry and redox biology as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.

Identification and characterization of thiamine diphosphate-dependent lyases with an unusual CDG motif.

The thiamine diphosphate (ThDP)-binding motif, characterized by the canonical GDG(X)24-27N sequence, is highly conserved among ThDP-dependent enzymes. We investigated a ThDP-dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. We found that JanthE has a high substrate promiscuity accepting long aliphatic α-keto acids as donors. Sterically hindered aromatic aldehydes or non-activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in the cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. We further determined the long-sought genuine tetrahedral intermediates formed with pyruvate and 2-oxo-butyrate in the pre-decarboxylation states and unravel atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP-binding motif and unlock a family of lyases able to perform valuable carboligation reactions.

Mechanisms of Cysteine-Lysine Covalent Linkage-The Role of Reactive Oxygen Species and Competition with Disulfide Bonds.

Recently, a new naturally occurring covalent linkage was characterised, involving a cysteine and a lysine, bridged through an oxygen atom. The latter was dubbed as the NOS bond, reflecting the individual atoms involved in this uncommon bond which finds little parallel in lab chemistry. It is found to form under oxidising conditions and is reversible upon addition of reducing agents. Further studies have identified the bond in crystal structures across a variety of systems and organisms, potentially playing an important role in regulation, cellular defense and replication. Not only that, double NOS bonds have been identified and even found to be competitive in relation to the formation of disulfide bonds. This raises several questions about how this exotic bond comes to be, what are the intermediates involved in its formation and how it competes with other pathways of sulfide oxidation. With this objective in mind, we revisited our first proposed mechanism for the reaction with model electronic structure calculations, adding information about the reactivity with alternative reactive oxygen species and other potential competing products of oxidation. We present a network with more than 30 reactions which provides one of the most encompassing pictures for cysteine oxidation pathways to date.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/ß Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2ß2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and ß, to initiate, using its metallo-cofactor in ß2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(ß) and Y731(α) across the α/ß interface. Using 2,3,5-F3Y122-ß2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-ß2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

N-hydroxypipecolic acid-induced transcription requires the salicylic acid signaling pathway at basal SA levels.

Systemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the SA induction-deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a KD of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for a slight NHP-mediated increase in NPR1 protein levels and NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signaling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic.

Structural basis for antibiotic action of the B1 antivitamin 2'-methoxy-thiamine.

The natural antivitamin 2'-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2'-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential.

Hydrazides Are Potent Transition-State Analogues for Glutaminyl Cyclase Implicated in the Pathogenesis of Alzheimer's Disease.

Amyloidogenic plaques are hallmarks of Alzheimer's disease (AD) and typically consist of high percentages of modified Aß peptides bearing N-terminally cyclized glutamate residues. The human zinc(II) enzyme glutaminyl cyclase (QC) was shown in vivo to catalyze the cyclization of N-terminal glutamates of Aß peptides in a pathophysiological side reaction establishing QC as a druggable target for therapeutic treatment of AD. Here, we report crystallographic snapshots of human QC catalysis acting on the neurohormone neurotensin that delineate the stereochemical course of catalysis and suggest that hydrazides could mimic the transition state of peptide cyclization and deamidation. This hypothesis is validated by a sparse-matrix inhibitor screening campaign that identifies hydrazides as the most potent metal-binding group compared to classic Zn binders. The structural basis of hydrazide inhibition is illuminated by X-ray structure analysis of human QC in complex with a hydrazide-bearing peptide inhibitor and reveals a pentacoordinated Zn complex. Our findings inform novel strategies in the design of potent and highly selective QC inhibitors by employing hydrazides as the metal-binding warhead.

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases.

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the "classical" SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.

ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.

Theoretical Studies of the Electronic Absorption Spectra of Thiamin Diphosphate in Pyruvate Decarboxylase.

Electronic absorption spectra are oftentimes used to identify reaction intermediates or substrates/products in enzymatic systems, as long as absorption bands can be unequivocally assigned to the species being studied. The latter task is far from trivial given the transient nature of some states and the complexity of the surrounding environment around the active site. To identify unique spectral fingerprints, controlled experiments with model compounds have been used in the past, but even these can sometimes be unreliable. Circular dichroism (CD) and ultraviolet-visible spectra have been tools of choice in the study of the rich chemistry of thiamin diphosphate-dependent enzymes. In this study, we focus on the Zymomonas mobilis pyruvate decarboxylase, and mutant analogues thereof, as a prototypical representative of the thiamin diphosphate (ThDP) enzyme superfamily. Through the use of electronic structure methods, we analyze the nature of electronic excitations in the cofactor. We find that all the determining CD bands around the 280-340 nm spectral range correspond to charge-transfer excitations between the pyrimidine and thiazolium rings of ThDP, which, most likely, is a general property of related ThDP-dependent enzymes. While we can confirm the assignments of previously proposed bands to chemical states, our calculations further suggest that a hitherto unassigned band of enzyme-bound ThDP reports on the ionization state of the canonical glutamate that is required for cofactor activation. This finding expands the spectroscopic "library" of chemical states of ThDP enzymes, permitting a simultaneous assignment of both the cofactor ThDP and the activating glutamate. We anticipate this finding to be helpful for mechanistic analyses of related ThDP enzymes.

Identification, characterization, and structure analysis of the cyclic di-AMP-binding PII-like signal transduction protein DarA.

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3'3'-cGAMP) but not c-di-GMP or 2'3'-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.

Converting Transaldolase into Aldolase through Swapping of the Multifunctional Acid-Base Catalyst: Common and Divergent Catalytic Principles in F6P Aldolase and Transaldolase.

Transaldolase (TAL) and fructose-6-phosphate aldolase (FSA) both belong to the class I aldolase family and share a high degree of structural similarity and sequence identity. The molecular basis of the different reaction specificities (transferase vs aldolase) has remained enigmatic. A notable difference between the active sites is the presence of either a TAL-specific Glu (Gln in FSA) or a FSA-specific Tyr (Phe in TAL). Both residues seem to have analoguous multifunctional catalytic roles but are positioned at different faces of the substrate locale. We have engineered a TAL double variant (Glu to Gln and Phe to Tyr) with an active site resembling that of FSA. This variant indeed exhibits aldolase activity as its main activity with a catalytic efficiency even larger than that of authentic FSA, while TAL activity is greatly impaired. Structural analysis of this variant in complex with the dihydroxyacetone Schiff base formed upon substrate cleavage identifies the introduced Tyr (genuine in FSA) to catalyze protonation of the central carbanion-enamine intermediate as a key determinant of the aldolase reaction. Our studies pinpoint that the Glu in TAL and the Tyr in FSA, although located at different positions at the active site, similarly act as bona fide acid-base catalysts in numerous catalytic steps, including substrate binding, dehydration of the carbinolamine, and substrate cleavage. We propose that the different spatial positions of the multifunctional Glu in TAL and of the corresponding multifunctional Tyr in FSA relative to the substrate locale are critically controlling reaction specificity through either unfavorable (TAL) or favorable (FSA) geometry of proton transfer onto the common carbanion-enamine intermediate. The presence of both potential acid-base residues, Glu and Tyr, in the active site of TAL has deleterious effects on substrate binding and cleavage, most likely resulting from a differently organized H-bonding network. Large-scale motions of the protein associated with opening and closing of the active site that seem to bear relevance for catalysis are observed as covalent intermediates are exclusively observed in the "closed" conformation of the active site. Pre-steady-state kinetics are used to monitor catalytic processes and structural transitions and to refine the kinetic framework of TAL catalysis.

Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.

Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot.

Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.

Observation of a stable carbene at the active site of a thiamin enzyme.

Carbenes are highly reactive chemical compounds that are exploited as ligands in organometallic chemistry and are powerful organic catalysts. They were postulated to occur as transient intermediates in enzymes, yet their existence in a biological system could never be demonstrated directly. We present spectroscopic and structural data of a thiamin enzyme in a noncovalent complex with substrate, which implicate accumulation of a stable carbene as a major resonance contributor to deprotonated thiamin.

Phosphate ions and glutaminyl cyclases catalyze the cyclization of glutaminyl residues by facilitating synchronized proton transfers.

Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization.